Polymorphic forms of 4-phenylamino quinazoline derivative, the preparation method and uses thereof

ABSTRACT

The present invention relates to polymorphic forms of N-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamide p-toluenesulfonate with the characteristic X-ray powder diffraction data as stated in the description, preparation methods thereof, pharmaceutical compositions comprising the same and the use thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/680,330, filed on Nov. 19, 2012, now U.S. Pat. No. 8, 937,079,which is a divisional application of U.S. application Ser. No.12/934,478, filed on Oct. 26, 2010, now U.S. Pat. No. 8,338,438, whichclaims priority to PCT Application No. PCT/CN2009/000317, filed on Mar.25, 2009, which claims priority from Chinese Patent Application200810043189.7, filed on Mar. 25, 2008, the disclosures and contents ofwhich are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to the polymorphic forms of 4-phenylaminoquinazoline derivatives. Specifically, the present invention relates tothe polymorphic forms of p-Toluenesulfonate salt of the compound offormula (I) (the chemical name isN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamide),their preparation methods, pharmaceutical compositions comprising saidpolymorphic forms and their use.

BACKGROUND OF THE INVENTION

Protein tyrosine kinase (PTK) is a type of enzyme which plays animportant role in normal cell growth. Protein tyrosine kinases catalyzethe transfer of phosphate groups from ATP to the residues of the proteinsubstrate. Many epidermal growth factor receptors (EGFR) have the effectof PTK, and the interaction of these receptors and growth factors isnecessary in normal cell growth regulation. However, the overexpressionof EGFR may cause excessive cell proliferation by the tyrosine kinaseaction of its own, and finally lead to the formation of tumors.

The epidermal growth factor receptor family can be divided into EGFR(Erb-BI), Erb-B2 (HER-2/neu), Erb-B3 and Erb-B4 according to structure.All of these epidermal growth factor receptors have been alreadyconfirmed to be related to most cancers.

Due to the important effect of the abnormal receptor kinases on thepathogenesis of cancer, the recent researches on anti-cancer agentsfocus on the development of the specific PTK inhibitors as potentialanti-cancer therapeutic agents. The research of quinazoline derivativesas PTK inhibitors for further application to cancer treatment arouseswide attraction.

WO 96/30347 (Chinese patent application CN 96102992) and WO 96/33980relate to some 4-(substituted-phenylamino)-quinazoline derivatives,their prodrugs, their pharmaceutically acceptable salts and their use intreating diseases caused by excessive cell proliferation.

WO 99/06378, WO 2000/31048 and WO 2000/06555 (Chinese patent applicationCN 99808949) also relate to substituted quinazoline derivatives havingirreversible PTK inhibitory activity.

WO 2006/071017 mentions some quinazoline derivatives that inhibit growthof cancer cells.

WO 2007/082434 describes a novel type of 4-phenylamino quinazolinederivatives and their use as PTK inhibitors, wherein, it is proven byexperiments that the compoundN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamideprepared in Example 8 possesses the relatively good effect of inhibitingthe growth of human epidermoid squamous cancer cell A431 and humanbreast cancer cell BT-474. The compound also possesses the significanttumor-inhibiting effect on human epidermoid squamous cancer cell A431implanted into a nude mouse. It is also proven by in vitro experimentsthat the compound has excellent inhibitory activity against Erb-B2kinase.

A crystalline form has some influence on the physical properties ofcompounds. Because of different crystal lattice structures,pharmaceutical compounds with many kinds of crystalline forms may havenot only different appearances (color and shape such as needle-shapedcrystal, crystalline lamellar and crystalline granule), but alsodifferent physical properties (such as melting point, solubility,density, stability and hygroscopicity), resulting in that they showdifferent dissolution and absorption behaviors in vivo, and this mighthave influence on the clinical effect and safety of the pharmaceuticalcompounds to a certain extent.

Specific crystalline form will have different thermodynamic behaviors ascompared to amorphous state or another crystalline form. Melting pointapparatus, thermogravimetric analysis (TGA) or Differential Scanningcalorimetry (DSC) and the like can be used in laboratories to measurethe thermal properties to differentiate a certain specific crystallineform, amorphous state and another crystalline form. Specific crystallineforms may have special spectral properties. For example, the data ofboth X-ray powder diffraction pattern and IR spectra can characterizespecific crystalline forms.

The above-mentioned documents are hereby fully incorporated in thisdisclosure by reference herein.

CONTENTS OF THE INVENTION

The present invention provides novel polymorphic forms ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamide(as defined in formula (I) below) p-Toluenesulfonate, their preparationmethods, pharmaceutical compositions comprising said polymorphic formsand their use in the manufacture of a medicament for the treatmentand/or prevention of tumors and the method of treatment and/orprevention of tumors in a mammal.

In summary, the present invention provides the technical solutions asfollows:

1. A crystalline p-Toluenesulfonate salt of the compoundN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamideas shown by formula (I):

2. A Crystalline form A of theN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate salt, i.e., Form A, characterized in that the X-raypowder diffraction pattern has peaks at the diffraction angle 2θ(°) of5.92±0.10, 8.64±0.10, 11.86±0.10, 16.58±0.10, 16.94±0.10, 17.86±0.10,19.12±0.10, 19.66±0.10, 20.12±0.10, 23.42±0.10, 24.14±0.10, 24.80±0.10,and 26.76±0.10.

3. Form A according to the technical solution 2, characterized in thatthe X-ray powder diffraction pattern provided by said Form A alsocontains peaks at the diffraction angle 2θ(°) of 9.80±0.10, 13.28±0.10,14.78±0.10, 17.36±0.10, 18.62±0.10, 21.62±0.10, 22.12±0.10, 22.38±0.10,23.14±0.10, 25.20±0.10, 27.24±0.10, 28.34±0.10, 28.78±0.10, 33.12±0.10,and 41.70±0.10.

4. Form A according to the technical solution 2 or 3, characterized inthat said Form A has the X-ray powder diffraction pattern assubstantially shown in FIG. 1.

5. Form A according to the technical solution 4, characterized in thatsaid Form A also has the IR spectrum as substantially shown in FIG. 5.

6. A process for preparing Form A according to any one of the technicalsolutions 2-5, comprising the steps of:

(a) dissolving the compound as shown in the following formula (I) in anorganic solvent to form a solution;

(b) adding dropwise a solution formed from p-Toluenesulfonic acid insaid organic solvent under stirring and controlling the molar ratio ofthe compound of formula (I) to p-Toluenesulfonic acid in the range of1:1˜6, preferably 1:3˜6; and

(c) resulting in a crystalline precipitate, and filtering out andwashing the crystal to obtain the target crystal.

7. A process according to the technical solution 6, characterized inthat the organic solvent is selected from the group consisting oftetrahydrofuran, methanol, ethanol, propanol, isopropanol, butanol,acetone, acetonitrile, DMSO, DMF, propanediol and the mixtures thereof,preferably consisting of tetrahydrofuran, methanol, ethanol, the mixtureof tetrahydrofuran and methanol, and the mixture of tetrahydrofuran andethanol.

8. A process according to the technical solution 7, wherein the organicsolvent is the mixture of tetrahydrofuran and methanol with a volumeratio of 1:1˜3, preferably 1:2˜3.

9. A process according to any one of the technical solutions 6-8,characterized in that the solution of the compound of formula (I) in thestep (a) has a concentration of 3˜8 g/100 mL, preferably 4˜6 g/100 mL.

10. A process according to any one of the technical solutions 6-9,characterized in that the solution of p-Toluenesulfonic acid in the step(b) has a concentration of 10˜30 g/100 mL, preferably 15˜25 g/100 mL.

11. Crystalline form B of theN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate salt, i.e., Form B, characterized in that the X-raypowder diffraction pattern has peaks at the diffraction angle 2θ(°) of4.72±0.10, 17.04±0.10, 19.32±0.10, and 24.12±0.10.

12. Form B according to the technical solution 11, characterized in thatthe X-ray powder diffraction pattern also has peaks at the diffractionangle 2θ(°) of 7.92±0.10, 9.54±0.10, 11.90±0.10, 12.94±0.10, 14.34±0.10,15.32±0.10, 17.88±0.10, 20.00±0.10, 21.80±0.10, 22.42±0.10, 25.08±0.10,25.80±0.10, 27.28±0.10, 28.00±0.10, and 28.44±0.10.

13. Form B according to the technical solution 11 or 12, characterizedin that said Form B has the X-ray powder diffraction pattern assubstantially shown in FIG. 2.

14. Form B according to the technical solution 13, characterized in thatsaid Form B also has the IR spectrum as substantially shown in FIG. 6.

15. A process for preparing Form B according to any one of the technicalsolutions 11-14, comprising the steps of:

(a) dissolving the crystalline form A ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate according to any one of the technical solutions 2-5in the mixture of a protic solvent and an aprotic solvent by heating toform a solution;

(b) maintaining the solution for 1-2h at the temperature of 40-80° C.;

(c) cooling the solution and resulting in a crystalline precipitate,letting the resulting mixture stand, filtering out and washing thecrystal to obtain the target crystal.

16. A process according to the technical solution 15, wherein the proticsolvent is water or the mixture of water and an alcohol.

17. A process of the technical solution 15 or 16, wherein the aproticsolvent is selected from the group consisting of tetrahydrofuran, ether,dichloromethane, acetone, acetonitrile, DMF, and the mixtures thereof.

18. A process according to any one of the technical solutions 15-17,wherein the solution of the crystalline form A of theN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate in the step (a) has a concentration of 2˜10 g/100 mL,preferably 4˜8 g/100 mL.

19. A process according to any one of the technical solutions 15-18,characterized in that the volume ratio of the protic solvent to theaprotic solvent in the mixture of a protic solvent and an aproticsolvent is 1:2˜4, preferably 1:3˜4.

20. Crystalline form C of theN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate salt, i,e., Form C, characterized in that the X-raypowder diffraction pattern has peaks at the diffraction angle 2θ(°) of3.40±0.10, 6.82±0.10, 7.58±0.10, 11.30±0.10, 14.84±0.10, 15.24±0.10,17.28±0.10, 17.86±0.10, 18.34±0.10, 20.32±0.10, 22.96±0.10, 23.50±0.10,24.12±0.10, 24.62±0.10, and 25.86±0.10.

21. Form C according to the technical solution 20, characterized in thatthe X-ray powder diffraction pattern also has peaks at the diffractionangle 2θ(°) of 9.04±0.10, 10.26±0.10, 22.44±0.10, 25.06±0.10,26.98±0.10, 28.62±0.10, and 29.98±0.10.

22. Form C according to the technical solution 20 or 21, characterizedin that said Form C has the X-ray powder diffraction pattern assubstantially shown in FIG. 3.

23. Form C according to the technical solution 22, characterized in thatsaid Form C also has the IR spectrum as substantially shown in FIG. 7.

24. A process for preparing Form C according to any one of the technicalsolutions 20-23, comprising the steps of:

(a) dissolving the crystalline form A ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate according to any one of the technical solutions 2-5in the mixture of a protic solvent and an aprotic solvent;

(b) adding dropwise a solution formed from p-Toluenesulfonic acid insaid mixture of a protic solvent and an aprotic solvent under stirringand controlling the molar ratio of said Form A to p-Toluenesulfonic acidin the range of 1:1˜6, preferably 1:3˜6; and

(c) resulting in a crystalline precipitate, letting the resultingmixture stand, and filtering out and washing the crystal to obtain thetarget crystal.

25. A process according to the technical solution 24, wherein the proticsolvent is water or the mixture of water and an alcohol.

26. A process of the technical solution 24 or 25, wherein the aproticsolvent is selected from the group consisting of tetrahydrofuran, ether,dichloromethane, acetone, acetonitrile, DMF, and the mixtures thereof.

27. A process according to any one of the technical solutions 24-26,wherein the solution of the crystalline form A of theN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate in the step (a) has a concentration of 3-7 g/100 mL,preferably 4-6 g/100 mL.

28. A process according to any one of the technical solutions 24-27,wherein the solution of p-Toluenesulfonic acid in the step (b) has aconcentration of 10-30 g/100 mL, preferably 15-25 g/100 mL.

29. A process of any one of the technical solutions 24-28, characterizedin that the volume ratio of the protic solvent to the aprotic solvent insaid mixture of a protic solvent and an aprotic solvent is 1:1˜5,preferably 1:3˜4.

30. A pharmaceutical composition comprising the crystalline formaccording to any one of the technical solutions 1-5, 11-14 and 20-23 anda pharmaceutically acceptable carrier.

31. A pharmaceutical composition of the technical solution 30, whichfurther comprises one or more additional drugs for the treatment and/orprevention of tumors.

32. Use of the crystalline form according to any one of the technicalsolutions 1-5, 11-14 and 2Q-23 in the preparation of a medicament forthe treatment and/or prevention of tumors.

33. A method for treating and/or preventing tumors in a mammal,comprising the administration of the crystalline form according to anyone of the technical solutions 1-5, 11-14 and 20-23 in an amounteffective in treatment and/or prevention to a mammal in need.

34. The use of the technical solution 32 or the method of the technicalsolution 33, wherein the tumor is selected from the group consisting ofbreast cancer, non-small cell lung cancer, ovarian cancer, gastriccancer, colonic cancer, pancreatic cancer and epidermatoid squamouscancer.

Specifically, in accordance with the first aspect of the presentinvention, there is provided the crystal of p-Toluenesulfonate salt ofcompound of formula (I), and also provided three polymorphic forms ofp-Toluenesulfonate salt of compound of formula (I), respectively namedas Form A, Form B and Form C.

Firstly, the present invention provides a crystal with the X-ray powderdiffraction pattern containing high intensity peaks at the diffractionangle 2θ(°) of 5.92±0.10, 8.64±0.10, 11.86±0.10, 16.58±0.10, 16.94±0.10,17.86±0.10, 19.12±0.10, 19.66±0.10, 20.12±0.10, 23.42±0.10, 24.14±0.10,24.80±0.10, and 26.76±0.10, and it is defined as Form A in the presentapplication.

In addition to the characteristic high strength diffraction peaks asmentioned above, the X-ray powder diffraction pattern of said Form Aalso contains intermediate strength peaks at the diffraction angle 2θ(°)of 9.80±0.10, 13.28±0.10, 14.78±0.10, 17.36±0.10, 18.62±0.10,21.62±0.10, 22.12±0.10, 22.38±0.10, 23.14±0.10, 25.20±0.10, 27.24±0.10,28.34±0.10, 28.78±0.10, 33.12±0.10, and 41.70±0.10.

The X-ray powder diffraction pattern of said Form A is substantiallyshown in FIG. 1. The relative strength of each peak is described in thefollowing table:

2θ(⁰) I/I₀ 2θ(⁰) I/I₀ 2θ(⁰) I/I₀ 2θ(⁰) I/I₀ 5.92 vs 8.64 vs 9.80 m 10.24w 11.86 s 13.28 m 14.78 m 15.36 w 16.58 vs 16.94 s 17.36 m 17.86 s 18.62m 19.12 s 19.66 s 20.12 vs 21.62 m 22.12 m 22.38 m 23.14 m 23.42 s 24.14s 24.80 s 25.20 m 25.90 w 26.76 s 27.24 m 28.34 m 28.78 m 30.14 w 32.06w 33.12 m 34.94 w 37.58 w 38.94 w 41.70 m 42.74 w 44.22 w

Form A also has the IR spectrum as substantially shown in FIG. 5.

Form A is yellow-green crystalline powder, with a melting point of 245°C.

In addition, the present invention also provides another crystal withthe X-ray powder diffraction pattern containing high strength peaks atthe diffraction angle 2θ(°) of 4.72±0.10, 17.04±0.10, 19.32±0.10, and24.12±0.10, and it is defined as Form B in the present application.

In addition to the characteristic high intensity diffraction peaks asmentioned above, the X-ray powder diffraction pattern of said Form Balso contains intermediate intensity peaks at the diffraction angle2θ(°) of 7.92±0.10, 9.54±0.10, 11.90±0.10, 12.94±0.10, 14.34±0.10,15.32±0.10, 17.88±0.10, 20.00±0.10, 21.80±0.10, 22.42±0.10, 25.08±0.10,25.80±0.10, 27.28±0.10, 28.00±0.10, and 28.44±0.10.

The X-ray powder diffraction pattern of Form B is substantially shown inFIG. 2. The relative intensity of each peak is described in thefollowing table:

2θ(⁰) I/I₀ 2θ(⁰) I/I₀ 2θ(⁰) I/I₀ 2θ(⁰) I/I₀ 4.72 vs 7.92 m 8.46 w 9.54 m11.90 m 12.26 w 12.94 m 14.34 m 15.32 m 17.04 s 17.88 m 18.50 w 19.32 s20.00 m 20.56 w 21.80 m 22.42 m 24.12 s 25.08 m 25.80 m 26.80 w 27.28 m28.00 m 28.44 m 29.50 w 30.22 w 31.54 w 42.30 w 44.54 w

Form B also has the IR spectrum as substantially shown in FIG. 6.

Form B is a pale yellow crystalline powder, with a melting point of235.4° C.

In addition, the present invention also provides a crystal with theX-ray powder diffraction pattern containing the high intensity peaks atthe diffraction angle 2θ(°) of 3.40±0.10, 6.82±0.10, 7.58±0.10,11.30±0.10, 14.84±0.10, 15.24±0.10, 17.28±0.10, 17.86±0.10, 18.34±0.10,20.32±0.10, 22.96±0.10, 23.50±0.10, 24.12±0.10, 24.62±0.10, and25.86±0.10, and it is defined as Form C in the present application.

In addition to the characteristic high intensity diffraction peaks asmentioned above, the X-ray powder diffraction pattern of said Form Calso contains middle intensity peaks at the diffraction angle 2θ(°) of9.04±0.10, 10.26±0.10, 22.44±0.10, 25.06±0.10, 26.98±0.10, 28.62±0.10,and 29.98±0.10.

The X-ray powder diffraction pattern of Form C is substantially shown inFIG. 3. The relative intensity of each peak is described in thefollowing table:

2θ(⁰) I/I₀ 2θ(⁰) I/I₀ 2θ(⁰) I/I₀ 2θ(⁰) I/I₀ 3.40 vs 4.80 w 6.82 VS 7.58vs 9.04 m 10.26 m 11.30 s 12.44 w 12.90 w 14.04 w 14.84 s 15.24 s 17.28vs 17.86 vs 18.34 s 19.05 w 20.32 s 21.36 w 22.44 m 22.96 s 23.50 s24.12 s 24.62 vs 25.06 m 25.86 vs 26.98 m 28.62 m 29.98 m 31.44 w 33.02w 34.44 w 37.02 w 37.80 w 38.84 w 44.35 w 46.68 w

Form C also has the IR spectrum as substantially shown in FIG. 7.

Form C is a yellow crystalline powder, with a melting point of 244° C.

In present invention, the powder X-ray diffraction patterns of the threetypes of crystals mentioned above were obtained by known methods in theart with RIGAKUD/MNX2550VB/PC X ray diffractometer.

In the powder X-ray diffraction pattern, each peak was identified byBragg formula calculation, and the site of each peak was determined bythe diffraction angle 2θ(°). The division of the intensity of peak onlyreflects the similar size of peaks in each site. In the presentinvention, each crystalline form took the highest diffraction peak ofits peak height as the base peak which was defined as I₀, with therelative intensity as 100% (for example, the peak at 2θ(°) of 5.92 inFIG. 1 as the base peak of Form A, the peak at 2θ(°) of 4.72 in FIG. 2as the base peak of Form B, the peak at 2θ(°) of 25.86 in FIG. 3 as thebase peak of Form C). Other peaks had the ratio of their height to theheight of the base peak as relative intensity I/I₀. The definition ofrelative intensity of each peak was shown in the following table:

Relative intensity I/I₀ (%) Definition 50~100 Vs (very strong) 20~50 S(strong) 5~20 M (medium) 1~5 W (weak)

The IR spectra of the three crystals mentioned above were measured bythe known method in the art, using Shimazu FTIR-8400S infraredspectrophotometer in the mode of KBr pelleting. The melting points ofthe crystals mentioned above were measured by the known method in theart, using WRS-2A/2 microcomputer melting point instrument.

Because of error of instrument or the difference of operators, oneskilled in the art can understand the slight difference in theparameters which characterize physical properties of crystals.Therefore, the parameters mentioned above only aid to characterize thepolymorphs provided in the present invention, not regarded as thelimitations to the polymorphs provided in the present invention.

In accordance with the second aspect of the present invention, there isprovided a process for preparing the three polymorphic forms of thepresent invention.

Firstly, the present invention provides a process for preparing Form A,comprising the steps of:

-   -   (a) dissolving the compound of formula (I) in an organic solvent        to form a solution;

-   -   (b) adding dropwise a solution formed from p-Toluenesulfonic        acid in said organic solvent under stirring and controlling the        molar ratio of the compound of formula (I) to p-Toluenesulfonic        acid in the range of 1:1˜6, preferably 1:3˜6;    -   (c) resulting in a crystalline precipitate, and filtering out        and washing the crystal to obtain the target crystal, the        crystalline form A of        N-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamide        p-Toluenesulfonate salt.

The organic solvent mentioned above is selected from the groupconsisting of tetrahydrofuran, methanol, ethanol, propanol, isopropanol,butanol, acetone, acetonitrile, DMSO, DMF, propanediol and the mixturesthereof, preferably consisting of tetrahydrofuran, methanol, ethanol,the mixture of tetrahydrofuranand methanol, the mixture oftetrahydrofuran and ethanol, especially the mixture of tetrahydrofuranand methanol. The volume ratio of tetrahydrofuran to methanol in themixture has a certain effect on the crystallization, and a preferredvolume ratio is 1:1˜3, particularly 1:2˜3.

The solution of the compound of formula (I) in the above step (a) has aconcentration of 3˜8 g/100 mL, preferably 4˜6 g/100 mL.

The solution of p-Toluenesulfonic acid in the above step (b) has aconcentration of 10˜30 g/100 mL, preferably 15˜25 g/100 mL.

Generally, the above preparation process is carried out under thecondition of cooling, room temperature or heating. It is noteworthy thatthe selection of reaction temperature has some effect on the formationof different crystals, which is also known by one skilled in the art.The crystallization temperature used in present invention is between−10° C. to the boiling point of the solvent as used, preferably 0˜40° C.

In addition, the present invention provides a process for preparing theForm B, comprising the steps of:

-   -   (a) dissolving the above crystalline form A of the        N-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamide        p-Toluenesulfonate in the mixture of a protic solvent and an        aprotic solvent by heating to form a solution;    -   (b) maintaining the solution for 1˜2 h at the temperature of        40˜80° C.;    -   (c) cooling the solution and resulting in a crystalline        precipitate, letting the resulting mixture stand, and filtering        out and washing the crystal to obtain the target crystal, the        crystalline form B of        N-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamide        p-Toluenesulfonate.

The said protic solvent is water or the mixture of water and an alcohol,such as methanol or ethanol, and particularly preferred is water. Thesaid aprotic solvent is selected from the group consisting oftetrahydrofuran, ether, dichloromethane, acetone, acetonitrile, DMF, andthe mixtures thereof, preferably tetrahydrofuran.

The volume ratio of the protic solvent to the aprotic solvent in themixture of a protic solvent and an aprotic solvent is 1:2˜4, preferably1:3˜4.

The solution of Form A in the above step (a) has a concentration of 2˜10g/100 m preferably 4˜8 g/100 mL.

Usually, the cooling as mentioned above shall be carried out till thetemperature is under room temperature. The standing time as mentionedabove is most preferred when Form B is completely precipitated, and thebetter standing time can be readily determined by one skilled in the artthrough several experiments.

Also, the present invention provides a process for preparing Form C,comprising the steps of:

-   -   (a) dissolving the above crystalline form A of the        N-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamide        p-Toluenesulfonate in the mixture of a protic solvent and an        aprotic solvent to form a solution;    -   (b) adding dropwise a solution formed from p-Toluenesulfonic        acid in said mixture of a protic solvent and an aprotic solvent        under stirring and controlling the molar ratio of Form A to        p-Toluenesulfonic acid in the range of 1:1˜6, preferably 1:3˜6;        and    -   (c) resulting in a crystalline precipitate, letting the        resulting mixture stand, and filtering out and washing the        crystal to obtain the target crystal.

The said protic solvent is water or the mixture of water and an alcohol,such as methanol or ethanol, and especially preferred is water. Theaprotic solvent is selected from the group consisting oftetrahydrofuran, ether, dichloromethane, acetone, acetonitrile, DMF, andthe mixtures thereof, preferably tetrahydrofuran.

The volume ratio of the protic solvent to the aprotic solvent in themixture of a protic solvent and an aprotic solvent is 1:1˜5, preferably1:3˜4.

The solution of Form A in the above step (a) has a concentration of 3˜7g/100 mL, preferably 4˜6 g/100 mL.

The solution of p-Toluenesulfonic acid in the above step (b) has aconcentration of 10˜30 g/100 mL, preferably 15˜25 g/100 mL.

In the present invention, the term “protic solvent” refers to a type ofsolvent with hydroxyl (i.e., —OH), such as water, an alcohol, such asmethanol, ethanol, and so on, preferably water or the mixture of waterand an alcohol, such as methanol or ethanol, especially preferablywater. The term “aprotic solvent” refers to an organic solvent withouthydroxyl, such as tetrahydrofuran, ether, dichloromethane, acetone,acetonitrile, DMF, and the mixtures thereof, preferably tetrahydrofuran.

In accordance with the third aspect of the present invention, there isprovided pharmaceutical compositions comprising the crystal/polymorphicform according to the present invention and a pharmaceuticallyacceptable carrier. In addition, the present invention also provides amethod for the treatment and/or prevention of tumors in a mammal,comprising administering the crystal/polymorphic form of the presentinvention to a mammal which needs such treatment or prevention in anamount effective in treatment and/or prevention.

Said pharmaceutical composition may be administered to a mammal (e.g.,human) by oral, rectal, parenteral (e.g., intravenous, intramuscular orsubcutaneous), or topical route. When the pharmaceutical composition isused, the pharmaceutical composition comprising the crystal/polymorphicform of the present invention in an amount effective in treatment orprevention is administered to a mammal (e.g., human) which needs suchtreatment or prevention. The term “an amount effective in treatment orprevention” refers to an amount of the active compound sufficient tocause the biological or medical response in a mammal (e.g., human)sought by the veterinarian or clinical physician. Ordinary physician,veterinarian and clinical physician can easily determine the effectiveamount of the crystal/polymorphic form of the present invention for thetreatment or prevention of the indicated diseases, which is usually0.01˜20 mg/kg of a patient's body weight per day, preferably 0.1˜10mg/kgof a patient's body weight per day. More specifically, the daily dosagefor a person of a body weight of 60 kg is usually 1˜1000 mg, preferably20˜500 mg. It is certain that the specific dosage will depend upon anumber of factors such as administration route, age, gender, weight andhealth conditions of a patient, as well as the special conditions to betreated, all of which are well within the abilities of a skilledphysician. The term “mammal” used herein includes, but is not limitedto, cat, dog, rabbit, goat, sheep, mouse, rat, human and the like, andhuman is particularly preferred.

The pharmaceutical composition provided in the present invention mayfurther comprise one or more other agents for the treatment and/orprevention of tumors. Said other agents for the treatment and/orprevention of tumors may be selected from the drugs which act on thechemical structure of DNA, such as Cisplatin and the like, the drugshaving influence on the synthesis of nucleic acid such as Methotrexate(MTX), 5-Fluorouracil (5FU) and the like, the drugs which affect thetranscription of nucleic acid such as Adriamycin, Epirubicin,Aclacinomycin, Mitramycin and the like, the drugs which act on thesynthesis of tubulin such as paclitaxel, Vinorelbine and the like,aromatized enzyme inhibitors such as Aminoglutethimide, Lentaron,Letrozole, and Anastrozole and the like, and inhibitors of the cellsignal pathway such as Imatinib, Gefitinib, Erlotinib, and the like.

Both the crystal/polymorphic form and the pharmaceutical composition ofthe present invention can be formulated in solid dosage forms for oraladministration, including capsules, tablets, pills, powders, granules,dragees and the like. In such solid dosage forms, thecrystal/polymorphic form provided in the present invention can be mixedwith at least one conventional inert excipient (or carrier). The inertexcipient (or carrier) includes but is not limited to (a) fillers orsolubilizers, such as starch, lactose, sucrose, glucose, mannitol andsilicic acid; (b) binders, such as hydroxymethyl cellulose, alginate,gelatin, polyvinylpyrrolidone, sucrose and arabic gum; (c) humectants,such as glycerol; (d) disintegrants, such as agar, calcium carbonate,potato starch or cassava starch, alginic acid, some composite silicates,polyvinylpolypyrrolidone and sodium carbonate; (e) retarding solvents,such as paraffin; (f) absorption accelerators, such as quaternaryammonium compounds; (g) wetting agents, such as cetyl alcohol andglycerol monostearate; (h) adsorbents, such as kaolin; and (i)lubricants, such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycol, sodium dodecyl sulfate, or the mixtures thereof.Capsules, tablets and pills can also contain buffers.

The solid dosage forms such as tablets, dragees, capsules, pills andgranulas can be prepared with coating and shell materials, such asenteric coatings or other materials well known in the art. They maycontain an opacifier. Besides, the release of the active compound in thecomposition can be carried out at a certain part of the alimentary canalin a delayed manner. When necessary, the active compound can beformulated in microencapsulated form with one or more of the aboveexcipients.

The crystal/polymorphic form and the pharmaceutical composition of thepresent invention can also be formulated in a liquid dosage form fororal administration, including pharmaceutically acceptable emulsion,solution, suspension, syrup or tincture. In addition to the crystallineform provided in the present invention as an active compound, the liquiddosage form may also contain inert diluents commonly used in the art,such as water or other solvents, solubilizers and emulsifiers, such asethanol, isopropanol, ethyl carbonate, ethyl acetate, propanediol,1,3-butanediol, dimethylformamide and plant oils, in particular,cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil andsesame oil, or the mixtures thereof. Apart from these inert diluents,the composition can also comprise auxiliaries, such as wetting agents,emulsifing and suspending agents, sweeteners, flavoring agents andperfuming agents.

When the pharmaceutical composition of the present invention is presentas a suspension, the suspension may further comprise, in addition to thecrystal/polymorph provided in the present invention, suspending agents,such as ethoxylated isooctadecanol, polyoxyethylene sorbitol anddehydrated sorbitan ester, microcrystalline cellulose, aluminiummethoxide and agar, or the mixtures thereof, and the like.

The crystal/polymorphic form and the pharmaceutical composition of thepresent invention can also be formulated into a dosage form forparenteral injection, including physiologically acceptable sterileaqueous or nonaqueous solution, dispersion, suspension or emulsion, andsterile powder which can be redissolved into sterile injectable solutionor dispersion. Aqueous and nonaqueous carriers, diluents, solvents orexcipients can be used to prepare said sterile aqueous or nonaqueoussolution, dispersion, suspension or emulsion. Suitable aqueous andnonaqueous carriers, diluents, solvents or excipients include water,ethanol, a polyol and suitable mixtures thereof.

The crystal/polymorphic form and the pharmaceutical composition of thepresent invention can be formulated into a dosage form for topicaladministration, including ointment, powder, patch, spray and inhalant.The crystal/polymorphic form provided in the present invention can bemixed with physiologically acceptable carriers and any preservatives,buffers, or required propellants, if necessary, under sterile condition.

In another aspect, the crystal/polymorphic form provided in the presentinvention can be used in the preparation of medicaments for thetreatment and/or prevention of the diseases mediated by protein tyrosinekinases. Said diseases include tumors, especially malignant tumors, suchas breast cancer, non-small cell lung cancer, ovarian cancer, gastriccancer, colonic cancer, pancreatic cancer, epidermoid squamous cancerand the like.

The crystal/polymorphic form of the present invention may beadministered alone or in combination with other pharmaceuticallyacceptable therapeutic agents, especially with other anti-tumor drugs.The therapeutic agents include, but are not limited to, the drugs whichact on the chemical structure of DNA, such as Cisplatin and the like,the drugs which affect the synthesis of nucleic acid such asMethotrexate (MTX), 5-Fluorouracil (5FU) and the like, the drugs whichaffect the transcription of nucleic acid such as Adriamycin, Epirubicin,Aclacinomycin, Mitramycin and the like, the drugs which act on synthesisof tubulin such as Paclitaxel, Vinorelbine and the like, aromatizedenzyme inhibitors such as Aminoglutethimide, Lentaron, Letrozole,Anastrozole and the like, inhibitors of the cell signal pathway such asImatinib, Gefitinib, Erlotinib, and the like. The various ingredients tobe combined can be administered simultaneously or sequentially, and canbe administered either in a single formulation or in separateformulations. Such a combination includes not only the combination ofthe crystal/polymorphic form provided in the present invention with oneadditional active agent but also the combination of thecrystal/polymorphic form of the present invention with two or more otheractive agents.

The main advantages of the polymorphic forms of the present inventioninclude:

-   -   (a) The polymorphs of        N-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-yl}-acrylamide        p-Toluenesulfonate salt provided in the present invention are        not apt to absorb moisture, and have the relatively good thermal        stability and light stability;    -   (b) It is proved by experiments in vivo that the polymorphs        provided in the present invention possess excellent        bioavailability in animals;    -   (c) It is proved by experiments that the polymorphs provided in        the present invention possess the excellent anti-tumor activity;    -   (d) It is proved by experiments in vivo that the polymorphs        provided in the present invention possess low toxicity and high        safety of administration in animals.

From the above description of the method for the preparation ofpolymorphs, the polymorphs of the p-Toluenesulfonate of the compound offormula (I) are obtained in the present invention by use of thedifferent solubility of the p-Toluenesulfonate of the compound offormula (I) in different solvents, which achieves the goal of purifyingthe p-Toluenesulfonate of the compound of formula (I). This purificationmethod is easy to operate, and is suitable for industrial production ina large scale.

DESCRIPTION OF FIGURES

FIG. 1 shows the X-ray powder diffraction pattern of Form A of theN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate;

FIG. 2 shows the X-ray powder diffraction pattern of Form B of theN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate;

FIG. 3 shows the X-ray powder diffraction pattern of Form C of theN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate;

FIG. 4 shows the superposed pattern of the X-ray powder diffractionpatterns of Forms A, B and C of theN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate, wherein the diffraction peaks of Form A arerepresented by thick real lines, Form B by fine real lines, and Form Cby dashed lines;

FIG. 5 shows the IR spectrum of Form A of theN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate;

FIG. 6 shows the IR spectrum of Form B of theN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate;

FIG. 7 shows the IR spectrum of Form C of theN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate.

The present invention will now be further described by certain examples.These examples are only to illustrate the present invention, but are notintended to limit its protection scope. For the experimental methods inthe following examples for which the specific conditions are notindicated, the conventional conditions or the conditions suggested bythe manufacturers are followed. The materials as used are obtainedcommercially or may be easily obtained by the person skilled in the artaccording to the methods in known literatures. As used herein, theabbreviation THF represents tetrahydrofuran; DMSO represents dimethylsulfoxide; DMF represents dimethylformamide; PVPP representspolyvinylpolypyrrolidone; PVP represents polyvinylpyrrolidone; ig refersto intragastric administration, and iv refers to intravenous injection.Unless otherwise indicated, the amounts and percents are measured byweight.

EXAMPLE Example 1 Preparation ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamideStep A: Preparation of4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-6-nitro-quinazoline

1.20 g (5.7 mmol) of 4-chloro-6-nitro-quinazoline (prepared by referringto WO 2007/082434) and 1.37 g (5.6 mmol) of4-(3-fluoro-benzyloxy)-3-chloro-aniline (prepared by referring to WO2007/082434) were dissolved in 80 mL of isopropanol and refluxed for 3hours. A large amount of yellow solid was precipitated from the system,and was filtered. The filter cake was washed with a saturated sodiumbicarbonate solution till pH=8 and dried under vacuum to obtain 1.62 g(3.75 mmol) of yellow solid, which was identified as the compound4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-6-nitro-quinazoline witha yield of 67%.

¹H-NMR(400 MHz, CDCl₃): δ11.30(1H, br), 9.54-9.48(1H, m), 8.45-8.41(1H,m), 8.31-8.25(1H, m), 7.98-7.89(1H, m), 7.50-7.47(1H, m), 7.35-7.26 (1H,m), 7.05-6.96(1H, m), 6.90-6.80(2H, m), 7.74-7.60(2H, m), 4.84(2H, s).

Step B: Preparation of4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-6-amino-quinazoline

1.60 g (3.77 mmol) of compound4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-6-nitro-quinazolineprepared according to the method of Step A, 1.05 g (18.85 mmol, 5 eq) ofreduced Fe powders, 2 mL of glacial acetic acid and 40 mL of methanolwere added to a flask equipped with a refluxing condenser and refluxedfor 2.5 hours in a 85° C. oil-bath. The Fe powders were removed byfiltration. The filtrate was diluted with ethyl acetate and washedsequentially with sodium bicarbonate solution and water. The organicphase was dried and concentrated to obtain 900 mg (2.28 mmol) of ayellow solid, which compound was identified as4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-6-amino-quinazoline witha yield of 61%.

¹H-NMR(400 MHz, DMSO): δ9.32(1H, s), 8.31(1H, s), 8.04(1H, d, J=2.64Hz), 7.73(1H, dd, J=2.64 Hz, 8.80 Hz), 7.54-7.43(2H, m), 7.36-7.28(3H,m), 7.26-7.14(3H, m), 5.57(2H, br), 5.27(2H, s).

Step C: Preparation ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamide

1.2 g (3.04 mmol) of4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-6-amino-quinazolineprepared according to the method of Step B, 0.6 mL (4.58 mmol, 1.5 eq)of triethylamine, 0.28 mL (3.33 mmol, 1.1 eq) of acryloyl chloride and40 mL of THF were added under the cooling by an ice-bath. The reactiontemperature rose to room temperature slowly. The reaction was stoppedafter 3 hours. The resultant mixture were filtered, and the filter cakewas washed with water to neutral and dried to obtain 1.0 g (2.23 mmol)of a yellow solid, which compound was identified asN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidewith a yield of 67%. MS: 449. mp:222-225° C.

¹H-NMR(400 MHz, CDCl₃+DMSO): δ8.75(1H, s), 8.60-8.52(2H, m), 7.81(1H, d,J=2.44 Hz), 7.69(2H, s), 7.54(1H, dd, J=2.56 Hz, 8.92 Hz), 7.30-7.22(2H,m), 7.18-7.08(2H, m), 6.96-6.86(2H, m), 6.37(2H, d, J=5.86 Hz), 5.67(1H,t, J=5.86 Hz), 5.06(2H, s).

Example 2 Preparation of the Crystal of p-Toluenesulfonate ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]quinazolin-6-yl}-acrylamide

3 g (6.68 mmol) of the compoundN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamideprepared according to the method of Example 1 was dissolved in themixture of tetrahydrofuran and methanol (THF/CH₃OH=2/1, 30 mL), and 24mL of a solution of p-toluenesulfonic acid (1 eq, 1.27 g) in the mixtureof tetrahydrofuran and methanol (THF/CH₃OH=1/1) was added dropwise intothe system slowly, and then a large amount of yellow solid was slowlyprecipitated from the system after the addition was finished. The solidwas filtered, and washed with water and dried under vacuum to obtain 2.6g (4.19 mmol) of yellow crystalline powder with a yield of 63% and apurity of 92.5%.

Example 3 Preparation of Form A of p-Toluenesulfonate ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]quinazolin-6-yl}-acrylamide

3 g (6.68 mmol) of the compoundN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamideprepared according to the method of Example 1 was dissolved in themixture of tetrahydrofuran and methanol (THF/CH₃OH=1/1, 50 mL), and a 38mL solution of p-toluenesulfonic acid (6 eq, 7.62 g) in the mixture oftetrahydrofuran and methanol (THF/CH₃OH=1/1) was added dropwise into thesystem slowly, and then a large amount of green yellow solid was slowlyprecipitated from the system during the addition. The solid wasfiltered, and the filter cake was washed with water and dried undervacuum to obtain 2.93 g of green yellow crystalline powder with a yieldof 70%. The X-ray powder diffraction pattern of the obtained crystal wasshown in FIG. 1. The IR spectrum of the crystal was shown in FIG. 5. Themelting point of the crystal was 245° C. This crystalline form wasdefined as Form A in the present application.

Example 4 Preparation of Form A of p-Toluenesulfonate ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]quinazolin-6-yl}-acrylamide

3 g (6.68 mmol) of the compoundN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamideprepared according to the method of Example 1 was dissolved in themixture of tetrahydrofuran and methanol (THF/CH₃OH=1/3, 60 mL), and a 38mL solution of p-toluenesulfonic acid (6 eq, 7.62 g) in the mixture oftetrahydrofuran and methanol (THF/CH₃OH=1/1) was added dropwise into thesystem slowly, and then a large amount of green yellow solid was slowlyprecipitated from the system during the addition. The solid wasfiltered, and the filter cake was washed with water and dried undervacuum to obtain 2.52 g of green yellow crystalline powder with a yieldof 61%. The X-ray powder diffraction pattern of the obtained crystal wasshown in FIG. 1. The IR spectrum of the crystal was shown in FIG. 5. Themelting point of the crystal was 245° C. This crystalline form wasdefined as Form A in in the present application.

Example 5 Preparation of Form B of p-Toluenesulfonate ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]quinazolin-6-yl}-acrylamide

3 g (4.84 mmol) of Form A of p-Toluenesulfonate ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamideprepared according to the method of Example 3 or 4 was added to themixture of tetrahydrofuran and water (THF/H₂O=4/1, 70 mL), and theresultant mixture was slowly risen to a temperature of 65° C. which wascontinuously kept for 20 min and then cooled to the room temperatureslowly, and then stood at 2° C. for 16 h. The resultant mixture werefiltered, and the filter cake was washed with water and dried undervacuum to obtain 1.68 g pale yellow crystalline powder with a yield of56%. The X-ray powder diffraction pattern of the obtained crystal wasshown in FIG. 2. The IR spectrum of the crystal was shown in FIG. 6. Themelting point of the crystal was 235.4 ° C. This crystalline form wasdefined as Form B in the present application.

Example 6 Preparation of Form B of p-Toluenesulfonate ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]quinazolin-6-yl}-acrylamide

3 g (4.84 mmol) of Form A of p-Toluenesulfonate ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamideprepared according to the method of Example 3 or 4 was added in themixture of tetrahydrofuran and water (THF/H₂O+2/1, 70 mL), and theresultant mixture was slowly risen to a temperature of 65° C. which wascontinuously kept for 20 min and then cooled to the room temperatureslowly, and then stood at 2° C. for 16 h. The resultant mixture werefiltered, and the filter cake was washed with water and dried undervacuum to obtain 2.30 g pale yellow crystalline powder with a yield of76%. The X-ray powder diffraction pattern of the obtained crystal wasshown in FIG. 2. The IR spectrum of the crystal was shown in FIG. 6. Themelting point of the crystal was 235.4° C. This crystalline form wasdefined as Form B in the present application.

Example 7 Preparation of Form C of p-Toluenesulfonate ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]quinazolin-6-yl}-acrylamide

3 g (4.84 mmol) of Form A of p-Toluenesulfonate ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamideprepared according to the method of Example 3 or 4 was added in themixture of tetrahydrofuran and water (THF/H₂O=3/1, 60 mL), and a 38 mLsolution of p-toluenesulfonic acid (6 eq, 7.62 g) in the mixture oftetrahydrofuran and methanol (THF/CH₃OH=2/1) was added dropwise into thesystem slowly, and then a large amount of yellow solid was slowlyprecipitated from the system during the addition. The solid wasfiltered, and the filter cake was washed with water and dried undervacuum to obtain 2.85 g of yellow crystalline powder with a yield of95%. The X-ray powder diffraction pattern of the obtained crystal wasshown in FIG. 3. The IR spectrum of the crystal was shown in FIG. 7. Themelting point of the crystal was 244° C. This crystalline form wasdefined as Form C in the present application.

Example 8 Preparation Form C of p-Toluenesulfonate ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]quinazolin-6-yl}-acrylamide

3 g (4.84 mmol) of Form A of p-Toluenesulfonate ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamideprepared according to the method of Example 3 or 4 was added in themixture of tetrahydrofuran and water (THF/H₂O=4/1, 60 mL), and a 38 mLsolution of p-toluenesulfonic acid (6 eq, 7.62 g) in the mixture oftetrahydrofuran and methanol (THF/CH₃OH=2/1) was added dropwise into thesystem slowly, and then a large amount of yellow solid was slowlyprecipitated from the system during the addition. The solid wasfiltered, and the filter cake was washed with water and dried undervacuum to obtain 1.95 g of yellow crystalline powder with a yield of65%. The X-ray powder diffraction pattern of the obtained crystal wasshown in FIG. 3. The IR spectrum of the crystal was shown in FIG. 7. Themelting point of the crystal was 244° C. This crystalline form wasdefined as Form C in the present application.

Example 9 Thermal Stability Test

A small amount of Form A was taken to be placed under a high temperatureenvironment of 60° C. to be stored for a month. The purity of Form A wasdetected at 0, 10 and 30 days, which was based to deduce the thermalstability of Form A. The thermal stability of Form B and Form C werealso deduced by this method. The results of the test were shown in thefollowing table.

Form Form A Form B Form C Detect time (day) 0 10 30 0 10 30 0 10 30Purity (%) 98.64 98.64 98.64 99.26 99.21 99.11 99.03 97.08 97.55

The above results of the test indicate that stored under the hightemperature of 60° C., Form A and Form B almost do not change as to thepurity, but the purity of Form C slightly decreases. It is indicatedthat Form A and Form B provided in the present invention have theexcellent thermal stability, and Form C has the good thermal stability.

Example 10 Light Stability Test

A small amount of Form A was taken to be placed under the environment ofillumination with an intensity of 4500|x±500|x to be stored for a month.The purity of Form A was detected at 0, 10 and 30 days, which was basedto deduce to the light stability of Form A. The light stability of FormB and Form C were also deduced by this method. The results of the testwere shown in the following table.

Form Form A Form B Form C Detect time 0 10 30 0 10 30 0 10 30 Purity (%)98.64 98.16 97.24 99.26 99.26 99.24 99.03 98.54 98.42

The above results of the test indicate that stored under the environmentof illumination with an intensity of 4500|x±500|x, Form B almost doesnot change as to purity, but the purities of Form A and Form C slightlydecrease. It is indicated that Form B provided in the present inventionhas the excellent light stability, and Form A and Form C have the goodlight stability.

Example 11 Hygroscopicity Test

A small amount of Form A was taken to be placed under a high moistureenvironment of 92.5% to be stored for a month. The purity of Form A wasdetected at 0, 10 and 30 days, which was based to deduce thehygroscopicity of Form A. The hygroscopicities of Form B and Form C werealso deduced by this method. The results of the test were shown in thefollowing table.

Form Form A Form B Form C Detect time 0 10 30 0 10 30 0 10 30 Purity (%)98.64 98.62 98.62 99.26 99.25 99.24 99.03 98.27 98.08

The above results of the test indicate that stored under a high moistureenvironment of 92.5%, Form A and Form B almost do not change as topurity, but the purity of Form C slightly decreases. It is indicatedthat the Form A and Form B provided in the present invention are verystable under the environment of high moisture, and Form C iscomparatively stable under the environment of high moisture.

The results of the Examples 9-11 indicate that the polymorphs providedin the present invention are relatively stable under the environment ofhigh temperature, illumination or high moisture.

Example 12 Test on the Drug Absorption in SD Rat (Sprague Dawley Rat)

Intragastric administration (ig): 16 healthy SD rats, male, weighed200-250 g, and grouped into 4 groups randomly, were each administeredintragastrically with a drug:the compound (21.68 mg/kg) preparedaccording to Example 1, Form A, B or C of p-toluenesulfonate of thecompound (30 mg/kg). The blood samples were collected at 0.5, 1.0, 1.5,2.0, 3.0, 5.0, 7.0, 9.0, 12 and 24 hours after administration, whichwere then isolated to prepare the plasma. The concentration of the drugin the plasma was determined by means of liquid chromatography/massspectrometry, and the concentration-time curve was obtained.

The main pharmacokinetics parameters were shown in the following table:

Dosage C_(max) AUC_(0-t) Compound (mg/kg) T_(max)(h) (ng/mL) (ng · h/mL)T_(1/2)(h) Compound of 21.68 0.75 32 106 1.81 formula (I) Form A 30 2.83187 977 1.49 Form B 30 1.25 253 978 1.23 Form C 30 2.25 161 577 1.27

Intravenous injection (iv): 4 healthy SD rats, male and weighed 200-250g, were intravenously administered with p-toluenesulfonate of thecompound of formula (I) (5 mg/kg). The blood samples were collected at5min, 15min, 0.5, 1.5, 2.0, 3.0, 4.0, 5.0 and 7.0 hours afteradministration, which were then isolated to prepare the plasma. Theconcentration of the compound in the plasma was determined by means ofliquid chromatography/mass spectrometry, and the concentration-timecurve was obtained.

The main pharmacokinetics parameters were shown in the following table:

Dosage C_(max) AUC_(0-t) Compound (mg/kg) T_(max)(h) (ng/mL) (ng · h/mL)T_(1/2)(h) p-Toluenesulfonate 5 0.083 1745 1860 1.55

Corrected by dosage, calculated by AUC_(0−t), the compound of formula(I) obtained in Example 1 has an absolute bioavailability of ig of0.95%, and the absolute bioavailability of ig of Form A was 8.75%, andthe absolute bioavailability of ig of Form B was 8.76%, and the absolutebioavailability of ig of Form C was 5.17%.

Example 13 Tumor-Inhibitory Effect on the Human Epidermatoid SquamousCancer A431 Transplanted to the BALB/cA Nude Mouse

A well-developed solid tumor A431 was selected and cut into severaluniform bits of a size of 2-3 mm under sterile conditions, with one bitbeing inoculated subcutaneously to the right armpit of each of theBALB/cA nude mice using trocar. 7 days after inoculation, the mice weregrouped randomly and were intragastrically administrated through mouthfor 13 days continuously. The long axis (a) and the short axis (b) ofthe tumors were measured with a vernier caliper every 4 days. Accordingto the formula V=ab²/2, the tumor volume (mm³) could be calculated. Thetested animals were neck-off killed 23 days after the inoculation, andanatomized to obtain the tumors. The tumors were weighed, and the tumorinhibition rate was calculated.

The results are shown in the table below, which indicate that Form A ofp-toluenesulfonate of the compound of formula (I) has the significantinhibitory effect on the tumor.

Number of Weight of Weight of Tumor Dosage Administration animalsanimals (g) tumors (g) inhibition Groups (mg/kg) route Start End(without tumor) x ± SD rate % Solvent 25 mL/kg ig 7 7 22.40 ± 2.81 1.13± 0.18 0 control Form A 25 ig 5 5 21.58 ± 2.18 0.79 ± 0.20 29.99 50 ig 55 22.87 ± 3.96 0.69 ± 0.17 38.67 100 ig 5 5 22.13 ± 1.83 0.64 ± 0.2343.63

Tumor-Inhibitor Effect on the Human Ovarian Cancer SKQV-3 Transplantedto the BALB/cA Nude Mouse

A tumor SKOV-3 in vigorous growth period was selected and cut intoseveral uniform bits of a size of about 1.5 mm³, which was inoculatedsubcutaneously to the right armpit of the BALB/cA nude mice using trocarunder sterile conditions. The diameters of the transplanted tumors ofnude mice were measured with a vernier caliper. The animals were groupedrandomly when the tumors grew to a size of 80-100 mm³. The tested animalgroup were intragastrically administrated through mouth as mentionedabove once a day for 3 weeks continuously. The positive control drug MMC(Mitomycin) was intravenously administered once on the first day with adose of 5 mg/kg. The negative control group were administered with 0.5%CMC-Na (Carboxymethyl Cellulose Sodium) with a dose of 0.2 mL/per mouse.The long axis (a) and the short axis (b) of the tumors were measuredtwice every week and the nude mice were weighed at the same time.According to the formula V=ab²/2, the tumor volume (mm³) could becalculated, based on which the relative tumor volume (RTV) wascalculated (Calculation Formula: RTV=V_(t)/V₀, wherein V₀ represents thetumor volume measured at the grouping time of administration, and V_(t)represents the tumor volume measured each time). The relative tumorproliferation rate T/C (%) was chosen as the index to evaluate theanti-tumor activity, which can be calculated according to the followingformula:

T/C (%)=(T _(RTV) /C _(RTV))×100

T_(RTV): RTV of the treated group; C_(RTV): RTV of the negative controlgroup

The standard for evaluating the effectiveness: T/C (%)>60% meansineffective and T/C (%) 60% means effective.

The results are shown in the table below, which indicate that Form A ofp-toluenesulfonate of the compound of formula (I) has the significanttumor inhibitory effect.

Dosage, Number of Volume of tumor Administration Animals (mm³) Groupsroute Start End V₀ V₂₁ RTV T/C (%) 0.5% CMC- 0.2 mL/per ig 12 12 85 ± 35 638 ± 339 9.6 ± 5.4 Na mouse MMC 5 mg/kg iv 6 6 83 ± 13 258 ± 77 3.1 ±0.5 32.0 Form A 200 mg/kg ig 6 6 86 ± 13 303 ± 72 3.5 ± 0.8 36.9 100mg/kg ig 6 6 87 ± 41 345 ± 88 4.3 ± 1.3 45.0 50 mg/kg ig 6 6 79 ± 28 421± 89 5.1 ± 1.7 53.0

V₀ represents the tumor volume before the administration, and V₂₁represents the tumor volume after 3 weeks of continuous administration.

According to the experimental method mentioned above, the tumor-bearingnude mice, into which the human ovarian cancer SKOV-3, the human lungcancer Calu-3, and the human lung cancer A549 were transplantedrespectively, were intragastrically administrated with Form B (100mg/kg) of the present invention through mouth twice a day for 3 weekscontinuously. The relative tumor proliferation rate T/C as obtained was28.5%, 35.1% and 56.3%, respectively.

According to the experimental method mentioned above, the tumor-bearingnude mice, into which the human ovarian cancer SKQV-3, the human lungcancer Calu-3, and the human lung cancer A549 were transplantedrespectively, were intragastrically administrated with Form C (100mg/kg) of the present invention through mouth twice a day for 3 weekscontinuously. The relative tumor proliferation rate T/C as obtained was23.2%, 39.4% and 58.7%, respectively.

The Form B and Form C of the p-toluenesulfonate of the compound offormula (I) were indicated to also have the significant tumor inhibitoryeffects.

Example 14 Toxicity Test of Long-Term Administration

80 healthy SD rats, male and weighed 200-250 g, were grouped into 16groups randomly, and were each intragastrically administered with FormA, B or C of the p-toluenesulfonate of the compound of formula (I) or ablank solvent (Five test groups with the dose of 20, 50, 100, 500 or 800mg/(kg·day) and a solvent control group were set). The results afterfour weeks of the continuous administration indicated that: the ratsadministered with the dose of 20, 50, 100, 500 or 800 mg/(kg·day) ofForm A, B or C respectively showed no abnormality in physical signs,appearance, behaviors, activities or shape of dejecta, and had a normalfood intake, and the weight and weight increase of the rats werebasically similar to the solvent control group without the statisticaldifference. The inspection results of hematology, blood biochemistry,cardiogram, body temperature and urine showed that the variousinspection indexes were similar to those of the control group and allvaried in the normal ranges, indicating that the polymorphic forms ofp-toluenesulfonate of the compound of formula (I) possessed low toxicityand high safety of administration.

Example 15 Pharmaceutical Composition

The capsules containing the p-toluenesulfonate ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidewere prepared from the following components:

Form A 15 g Starch 15 g Lactose 30 g PVPP 2.5 g PVP 2.5 g Talcum powder3 g Sodium dodecyl sulfate 4 g

According to the conventional method, Form A of the p-toluenesulfonateofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamideand starch were mixed and sieved, and then evenly mixed with othercomponents mentioned above, and filled into common gelatin capsules.

Example 16 Pharmaceutical Composition

The tablets containing the p-toluenesulfonate ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidewere prepared from the following components:

Form B 20 g Starch 20 g Lactose 40 g PVPP 3 g PVP 3 g Talcum powder 1.6g Sodium dodecyl sulfate 5 g

According to the conventional method, Form B of p-toluenesulfonate ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamideand starch were mixed and sieved, and then evenly mixed with othercomponents mentioned above, and pressed into tablets directly.

All documents referred to throughout this application are hereby fullyincorporated by reference herein, just as each of them is individuallycited to be incorporated into this application. Further, it would beappreciated that, after reading the above contents of the presentinvention, the skilled person in the art may make various changes ormodifications to the invention without violating the spirit of thepresent invention, and these forms of changes would also fall within thescope defined in the claims of the application.

1. Crystalline form C of theN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate salt, characterized in that the X-ray powderdiffraction pattern has peaks at the diffraction angle 2θ(°) of3.40±0.10, 6.82±0.10, 7.58±0.10, 11.30±0.10, 14.84±0.10, 15.24±0.10,17.28±0.10, 17.86±0.10, 18.34±0.10, 20.32±0.10, 22.96±0.10, 23.50±0.10,24.12±0.10, 24.62±0.10, and 25.86±0.10.
 2. Form C according to claim 1,characterized in that the X-ray powder diffraction pattern also haspeaks at the diffraction angle 2θ(°) of 9.04±0.10, 10.26±0.10,22.44±0.10, 25.06±0.10, 26.98±0.10, 28.62±0.10, and 29.98±0.10.
 3. FormC according to claim 1, characterized in that the Form C has the X-raypowder diffraction pattern as substantially shown in FIG.
 3. 4. Form Caccording to claim 1, characterized in that the Form C also has the IRspectrum as substantially shown in FIG.
 7. 5. A process for preparing acrystalline form C of theN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate salt, comprising the steps of: (a) dissolving thecrystalline form A ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate in the mixture of a protic solvent and an aproticsolvent; (b) adding dropwise a solution formed from p-Toluenesulfonicacid in said mixture of a protic solvent and an aprotic solvent understirring and controlling the molar ratio of Form A to p-Toluenesulfonicacid in the range of 1:1˜6; (c) resulting in a crystalline precipitate,letting the resulting mixture stand, and filtering out and washing thecrystal to obtain the target crystal.
 6. A process according to claim 5,wherein the protic solvent is water or the mixture of water and analcohol.
 7. A process according to claim 5, wherein the aprotic solventis selected from the group consisting of tetrahydrofuran, ether,dichloromethane, acetone, acetonitrile, DMF, and the mixtures thereof.8. A process according to claim 5, wherein the solution of thecrystalline form A ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate in the step (a) has a concentration of 3˜7 g/100 mL.9. A process according to claim 5, wherein the solution ofp-Toluenesulfonic acid in the step (b) has a concentration of 10˜30g/100 mL.
 10. A process according to claim 5, characterized in that thevolume ratio of the protic solvent to the aprotic solvent in the mixtureof a protic solvent and an aprotic solvent is 1:1˜5.
 11. A processaccording to claim 5, wherein the solution of the crystalline form A ofN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate in the step (a) has a concentration of 3˜7 g/100 mL;the solution of p-Toluenesulfonic acid in the step (b) has aconcentration of 10˜30 g/100 mL; the protic solvent is water; theaprotic solvent is tetrahydrofuran, and the volume ratio oftetrahydrofuran to water is 1˜5:1.
 12. A pharmaceutical compositioncomprising the crystalline form C of theN-{4-[3-chloro-4-(3-fluoro-benzyloxy)phenylamino]-quinazolin-6-yl}-acrylamidep-Toluenesulfonate salt and a pharmaceutically acceptable carrier.